Opioid receptors belong the superfamily of G protein-coupled receptors (GPCRs). Similar to a majority of this superfamily members, prolonged activation of the opioid receptors resulted in a loss of response, mainly due to receptor desensitization. There is overwhelming evidence to suggest that the phosphorylation of GPCRs is the general mechanism for receptor desensitization. In the case of opioid receptor, phosphorylation of the mu- nd delta-opioid receptor upon agonist activation have been reported. Though there is some peripheral indication of a relationship between delta-opioid receptor phosphorylation and receptor desensitization, detailed correlation has not been established. In our studies with mu- opioid receptor phosphorylation, we could demonstrate that receptor phosphorylation occurred within minutes of agonist binding, while the ability of agonist to inhibit adenylyl cyclase was not blunted until hours after agonist addition. Therefore, we decided to investigate thoroughly the relationship between delta-opioid receptor phosphorylation and desensitization. We will utilize the polyclonal antibodies specific against the delta-opioid receptor and hemagglutinin (HA) epitope tagged receptor we have developed in our studies. We will correlate the degree of delta opioid receptor phosphorylation/dephosphorylation to the ability of agonist in inhibiting the forskolin-stimulated adenylyl cyclase activity. We will investigate the effect of various protein kinases' inhibitors on receptors phosphorylation and desensitization. We will pin-point the phosphorylation sites on the delta-opioid receptor which are involved in receptor desensitization. This will be accomplish by the receptor truncational and mutational analysis. The attenuation of receptor phosphorylation with the removal of putative phosphorylation sites, SER and Thr, and the subsequent effect on agonist-induced receptor desensitization will be determined. In order to eliminate any misconclusion, effect on mutation of Ser/Thr of interest to Asp will be evaluated and the amino acid sequencing of the receptor domains involved in phosphorylation will be carried out. Finally, the protein kinases which re involved in opioid receptor phosphorylation will be identified by the transient expression of these kinases in HEK293 cells which stably expressing the delta-opioid receptor. The effect of the over-expression of these kinases,or the dominant mutants of GRK on receptor phosphorylation and desensitization will be determined. The probably presence of a specific kinases for delta-opioid receptor will be investigated by the in vitro phosphorylation and peptide mapping of the phosphorylated purified receptor carried out with endogenous and exogenous protein kinases.